The U.S. Department of State recently invited us to collaborate with Russian Research Center of Molecular Diagnostics and Therapy (RDCMDT) in the evaluation of nanoparticles as drug delivery vehicles for anti-tuberculosis drugs. While the project by RDCMDT is funded through the International Science and Technology Center (ISTC), funding for the portions of the project to be performed at National Jewish must be funded from U.S. domestic sources. Nanoparticles are small (Submicron) colloidal particles in which different drugs can be entrapped or adsorbed, and their usefulness as an advanced drug delivery system has been demonstrated in many areas. They can be stored without refrigeration for extended periods, and may enable targeting of anti-tuberculosis drugs to macrophages, and the intracellular compartments of macrophages in which Mycobacterium tuberculosis resides. We hypothesize that formulations of existing antituberculosis drugs within nanoparticles modified to traffic to intracellular compartments bearing M. tuberculosis will result in a reduction of the minimal inhibitory concentrations (MIC) relative to the free drugs. Our preliminary data, the only data on anti-TB drugs, suggest that the MICs for streptomycin and isoniazid were reduced by 4.5-7 fold. Dr. Geuelperina at RDCMDT has already begun encapsulation of ethambutol, pyrazinamide, capreomycin, amikacin, kanamycin, ethionamide, levofloxacin, cycloserine, moxifloxacin, vancomycin, and cefoxitin. These drugs, if efficiently encapsulated, will be tested in a macrophage model of M. tuberculosis infection at National Jewish Medical and Research Center in Denver. We will test the hypothesis that increasing the intracellular accumulation, particularly in phagosomes harboring M. tuberculosis will result in enhanced efficacy, as measured by reduced growth and viability of intracellular organisms. We will characterize the accumulation of each formulation by labelling the drugs and following their routes of uptake when the particles are opsonized by serum proteins, when opsonization is inhibited by coating the particles with polyethylene glycol, and when the nanoparticles are targeted to specific cell surface receptors via modification with transferrin or mannoside clusters. Distribution of the drugs within the cells over time will also be monitored by immunostaining of the drug molecules within the cells at increasing intervals following uptake. The immunostaining will be made quantitative by creating standard curves for each drug concentration, such that concentrations within particular cellular compartments can be estimated. Finally, we will correlate viability of intracellular M. tuberculosis with drug exposure at the sub cellular level, and determine which formulations are most effective at directing drug accumulation in the cellular compartments harboring M. tuberculosis. Successful completion of this project will provide us with the most promising formulations of encapsulated anti-TB drugs which will create a basis for further collaboration with RDCMDT in preclinical development, including animal models of efficacy, pharmacokinetics, and toxicology.